Methods for making and using a Chlorella extract having immunomodulating properties

ABSTRACT

Chlorella extracts containing high molecular weight Chlorella polysaccharide and polysaccharide complexes show immune modulatory, specifically immune stimulatory, activity. The polysaccharide and polysaccharide complexes contain glucose and any combination of: galactose, rhamnose, mannose and arabinose, as well as N-acetyl glucosamine and N-acetyl galactosamine. The extracts may be treated with pronase, DNAse, RNAse and proteases to remove unassociated nucleic acids, ribonucleic acids and proteins. The extracts may also undergo treatment to effect cleavage of specific glycosidic linkages, the linkages being defined by their susceptibility to cleavage by amylase, amyloglucosidase, cellulase or neuraminidase. Chlorella extracts may be administered to a mammal to increase proliferation of splenocytes and increase production of cytokines such as IL-6, IL-10, INF-γ and TNF-α. They may be used as a supplement to a vaccination regimen.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/224,014, filed Aug. 10, 2000, the content of which isherein incorporated by reference.

FIELD OF INVENTION

[0002] The present invention relates to Chlorella extracts for use asimmunomodulators.

BACKGROUND OF THE INVENTION

[0003] Chlorella is a unicellular green algae that has been called asun-powered supernutrient. Attested beneficial properties of this ediblemicroalgae include wound healing, detoxification, constipation reliefand growth stimulation. A number of studies have also indicated thatChlorella has beneficial effects on the immune system, both in vitro andin vivo.

[0004] Chlorella occurs in both fresh water and marine water. Species ofthe Chlorella genus exhibit striking diversity of physiological andbiochemical properties (Kessler, E. “Phycotalk” 1989, 1:141-153; V.Rastogi Publ., New Delhi, India). Chlorella produces little celluloseand other indigestible cell wall material, and hence has beenextensively investigated as a possible new source of food, especially asfeedstock (Lee, Robert E. “Phycology” 2^(nd) edition; 1989, page 281;Cambridge University Press).

[0005] Chlorella has the highest content of chlorophyll of any knownplant. It also contains vitamins, minerals, dietary fibre, nucleicacids, amino acids, enzymes, etc. It contains more than 9% fats (out ofwhich polyunsaturated fatty acids [PUFA] represent about 82%). Thevitamin content consists of provitamin A, vitamins B₁, B₂, B₆, niacin,B₁₂, biotin, vitamin C, vitamin K, pantothenic acid, folic acid,choline, lipoic acid, ionositol, PABA etc. Among the minerals presentthe most important are P, K, Mg, S, Fe, Ca, Mn, Cu, Zn and Co. The maincomponents of Chlorella cells are about 60% protein (composed of allbasic amino acids), and 20% carbohydrate.

[0006] Aqueous extracts of Chlorella pyreinodosa has been used for itsnutritive content as well as for other health benefits. Studies reporton numerous health benefits including improved immune system functionand detoxification of harmful toxins. It was introduced as a health foodin the USA in 1977 when novel technology processes were developed whichmade it more digestible and has been the largest selling health foodsupplement in Japan for a number of years. The Taiwan Chlorella companyis the world's largest supplier of Chlorella, and sells the productworldwide to Asia, Europe and North America, under the following brandnames: Algea, Bio-REU-RELLA, Green Gem, Green Boost, Green Nature, GreenPower, Joyau Vert and Natural Boost.

[0007] A number of studies have documented C. vulgaris extracts whichhave anti-tumor activity, as well as activity against Listeria and E.coli (Tanaka et al. Immunopharmacol. Immunotoxicol., 1990,12(2):277-291; Tanaka et al. Cancer Immunol. Immunother., 1998,45(6):313-320; Hasegawa et al. Int. J. Immunopharmacol., 1990,12(8):883-891). These activities appear to be immune-mediated ratherthan direct toxicity against the tumor or pathogen.

[0008] A number of Chlorella extracts are available commercially,including products by Swiss Herbal and Nature's Way. The Swiss Herbalproduct is identified as pure Chlorella broken cells containing Protein61%, Carbohydrate 21.1%, Fat 11.0%, Chlorophyll 2.866%, RNA 2.94% andDNA 0.28%.

[0009] Other publications related to the present field include thefollowing:

[0010] Japanese patent application laid open No. Sho 58-15920 disclosespolysaccharides from fresh water Chlorella having immune potentiator andanti-tumor activity.

[0011] Neveu et al. Experientia, 1978, 34(12):1644-1645 discloses thatC. pyrenoidosa is an immune response modulator.

[0012] Vermeil and Morin CR Seances Soc. Biol. Fil., 1976,170(3):646-649 discloses that C. pyrenoidosa, presumably by nature ofits cell wall, protects mice against sarcoma grafting.

[0013] Miyazawa et al. J. Ethnopharmacol., 1988, 24(2-3):135-146discloses that C. pyrenoidosa cells or extract mediate host immuneenhancement of the anti-tumor response.

[0014] Umezawa et al. Chemotherapy, 1982, 30(9):1041-1046 and Komiyamaet al. Chemotherapy, 1986, 34:302-307 disclose that the acidicpolysaccharide Chlon A from C. pyrenoidosa has immune enhancing andanti-tumor effects. Chlon A contains rhamnose, arabinose, glucose,galactose and glucuronic acid.

[0015] White and Barber Biochimica Biophysica Acta, 1972, 264:117-128discloses an 88 kDa acidic polysaccharide from C. pyrenoidosa containingmainly rhamnose, as well as arabinose, galactose, xylose, mannose andglucuronic acid.

[0016] U.S. Pat. No. 4,533,548 discloses acidic polysaccharide CH-1 fromC. pyrenoidosa containing mainly rhamnose, as well as arabinose,galactose, glucose and glucuronic acid. The polysaccharide was obtainedvia gel filtration with Sephadex G-75.

[0017] U.S. Pat. No. 4,831,020 discloses a polysaccharide extract fromC. minutissima, a marine Chlorella, with immune-stimulating andanti-tumor activity. This patent states that polysaccharides from marineChlorella species are more effective in activating immunity than freshwater Chlorella species. The polysaccharide extract was obtained via gelfiltration with Sephadex G-50.

[0018] U.S. Pat. No. 4,786,496 discloses a lipid and glycolipid fractionof marine Chlorella with immuno-potentiating activity.

[0019] Kojima et al. J. Retic. Sbc., 1973, 14:192-208 discloses a1,250-1400 Da reticuloendothelial system (RES)active glucan fromChlorella.

[0020] U.S. Pat. No. 3,462,412 discloses a process for preparing aRES-stimulating extract from Chlorella.

[0021] Japanese patent application, publication no. 06248003 discloses aChlorella extract of 15 to 25 kDa, comprising polysaccharides containingpredominantly galactose, with anti-neoplastic activity.

[0022] Mizuno et al. Bull. Fac. Agr. Shizuoka Univ. (Shizuoka DaigakuNogakubu Kenkyu Hokoku), 1980, 30:51-59 discloses two fractions ofneutral glycans from Chlorella, both apparently of small molecularweight.

[0023] Ukai et al. Ann. Proc. Gifu Pharm. Univ. (Gifu Yakka DaigakuKiyo), 1990, 39:44-48 discloses two polysaccharides, CP-I and CP-II,from C. pyrenoidosa with RES-stimulating activity. CP-I comprisesglucose, fucose, rhammose, galactose and mannose; CP-II comprisesglucose, galactose, rhamnose and mannose.

[0024] Chu et al. Aquaculture, 1982, 29(3-4):241-252 discloses that thepolysaccharide, ethanol-precipitable fraction of five algal speciesincluding Chlorella contains principally glucose, mannose,ribose/xylose, rhamnose and fucose.

SUMMARY OF THE INVENTION

[0025] Chlorella extracts prepared according to the invention showimmune stimulatory activity in pharmacological and clinical tests. Inone aspect, the extracts provided by the present invention have a higherimmune stimulatory activity than extracts prepared and used in the art.

[0026] In one aspect, the invention provides preparations comprisinghigh molecular weight Chlorella polysaccharide and polysaccharidecomplexes. The high molecular weight polysaccharide and polysaccharidecomplexes are about 1×10⁵ Da to about 1×10⁷ Da and constitute at least22% (w/w) of the total Chlorella-derived content of the extract. In apreferred embodiment, the extract is derived from Chlorella pyrenoidosa.

[0027] The high molecular weight polysaccharide and polysaccharidecomplexes may be of a selected range, e.g. about 1×10⁵ Da to about 3×10⁵Da, about 3×10⁵ Da to about 5×10⁵ Da, about 5×10⁵ Da to about 6×10⁵ Da,about 6×10⁵ Da to about 7×10⁵ Da, about 7×10⁵ Da to about 8×10⁵ Da,about 8×10⁵ Da to about 9×10⁵ Da, about 9×10⁵ Da to about 1×10⁶ Da,about 1×10⁶ Da to about 2×10⁶ Da, about 2×10⁶ Da to about 3×10⁶ Da,about 3×10⁶ Da to about 4×10⁶ Da, about 4×10⁶ Da to about 5×10⁶ Da,about 5×10⁶ Da to about 7×10⁶ Da, about 7×10⁶ Da to about 9×10⁶ Da, andabout 9×10⁶ Da to about 1×10⁷ Da.

[0028] The extracts may contain various different percentages ofpolysaccharide and polysaccharide complexes as a fraction of the totalChlorella-derived content of the extract. The percentage may be at least24% (w/w), at least 26% (w/w), at least 28% (w/w), at least 30% (w/w),at least 35% (w/w), at least 40% (w/w), at least 45% (w/w), at least 50%(w/w), or at least 60% (w/w).

[0029] In another aspect, the high molecular weight polysaccharide andpolysaccharide complexes contain glucose and any combination of:galactose, rhamnose, mannose and arabinose.

[0030] In another aspect, the high molecular weight polysaccharide andpolysaccharide complexes is substantially free of ribose, nucleic acids,ribonucleic acids and unassociated proteins. The high molecular weightpolysaccharide and polysaccharide complexes may also contain N-acetylglucosamine and N-acetyl galactosamine.

[0031] In another aspect, the extracts of the invention retainimmunomodulating activity upon treatment to remove unassociated DNA, RNAand proteins. Such treatment includes digestion by pronase, DNAse, RNAseand proteases.

[0032] In another aspect, the extracts of the invention retainimmunomodulating activity upon treatment to effect cleavage of specificglycosidic linkages, the linkages being defined by their susceptibilityto cleavage by amylase, amyloglucosidase, cellulase or neuraminidase.Such susceptible linkages are typically:

[0033] (i) three or more α-1,4-linked D-glucose units;

[0034] (ii) α-1,4-linked glucosides;

[0035] (iii) α-1,4-linked galactosides; or

[0036] (iv) α-1,4-linked D-glucose.

[0037] The invention also provides nutritional compositions containingthe high molecular weight Chlorella extract with at least one energysource which may be carbohydrates, fats or nitrogen.

[0038] The extracts of the invention may also be used in combination orin mixture with a conventional supplement such as vitamin E, vitamin Cand folic acid. The extracts may also be used with other nutraceuticalssuch as fish oils, spirulina and echinacea, especially thosenutraceuticals which have immunostimulant activity.

[0039] The invention also provides a process for obtaining Chlorellapreparations having immunomodulating activity. The process contains thesteps of:

[0040] (a) size fractionating an aqueous extract of Chlorella, and

[0041] (b) selecting fractions comprising high molecular weightpolysaccharide and polysaccharide complexes of about 1×10⁵ Da to about1×10⁷ Da.

[0042] The process for obtaining the Chlorella extract may furtherinclude the step of pooling and concentrating the selected fractions.Size fractionation may be achieved by chromatography, ultrafiltration orultracentrifugation.

[0043] The invention also provides a method for modulating the immuneresponse of a mammal including humans by administering to the mammal aneffective amount of the high molecular weight Chlorella extract. Suchmodulation includes increased proliferation of splenocytes and increasedproduction of cytokines such as IL-6, IL-10, INF-γ and TNF-α, and may beadvantageously used to treat or prevent bacterial or fungal infections.

[0044] The extract may further be administered as a supplement to avaccination regimen to further stimulate the immune response. A fluvaccine may be advantageously used with the extract. The extract may bepresent as an adjuvant to the vaccines, especially as an oral vaccineadjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The invention in its various embodiments will now be describedwith reference to the attached drawings.

[0046]FIG. 1: Combination size-exclusion chromatography (SEC)/refractiveindex detection (RI)/multi-angle laser light scattering (MALS)chromatogram of immune booster preparations IBP-1 or IBP-2. The toptrace is the RI chromatogram; the bottom trace is the MALS response;

[0047]FIG. 2: Cumulative molecular weight profile of IBP-1;

[0048]FIG. 3: SEC chromatogram of IBP-2 using Sephacryl S 1000 SF;

[0049]FIG. 4: SEC chromatogram of IBP-2 (or IBP-1) using Sephacryl S 300HR;

[0050]FIG. 5: Stimulation of splenocyte proliferation by variousChlorella extract fractions resolved by Sephacryl S 300 HR; IBP-6s isthe peak shoulder fraction of IBP-6; ‘Cells’ and ‘Cells+DMSO’ arenegative controls; LPS is lipopolysaccharide, used as a positivecontrol;

[0051]FIG. 6: Stimulation of splenocyte proliferation by variousChlorella extract fractions resolved by Sephadex G100 chromatography ofIBP-2; ‘Cells’ is a negative control; LPS is a positive control;

[0052]FIG. 7: Splenocyte proliferation—Titration curve of IBP-2; ‘Cells’is a negative control; LPS is a positive control;

[0053]FIG. 8: Proliferation assay measuring ³H-incorporation by isolatedB- or T-cells in the presence of 25 μg/mL IBP-2;

[0054]FIG. 9: Nitric oxide production by BALB/c inflammatory peritonealmacrophages cultured in the presence of various concentrations of IBP-2;‘IFN+LPS’ is a positive control;

[0055]FIG. 10: IL-6 production by BALB/c mouse spleen cells in thepresence of various concentrations of IBP-2; ‘Con A’ is Concanavalin A;‘Cells+ConA’ and ‘Cells+LPS’ are positive control samples, ConcanavalinA is at 10 μg/mL; LPS is at 20 μg/mL;

[0056]FIG. 11: Effect of IBP-2 compared to 4 mg echinacea (Echin.) onListeria monocytogenes proliferation in mice;

[0057]FIG. 12: Effect of IBP-2 compared to crude algae and echinacea onCandida albicans proliferation in mice;

[0058]FIG. 13: Mouse splenocyte proliferation as measured by³H-incorporation, cultured in the presence of IBP-2 or commercialChlorella extracts from Swiss Herbal and Nature's Way.

[0059]FIG. 14: Capillary electrophoresis chromatogram of the dialyzedcrude extract IBP-2. The monosaccharides are assigned to the peaks asfollows: ribose at position 9.05; rhamnose at position 10.47; mannose atposition 10.68; galactose at position 11.14; and glucose at position11.65.

[0060]FIG. 15: Capillary electrophoresis chromatogram of the retainedportion after IBP-2 was passed through a lMDa MWCO ultrafiltrationmembrane. The monosaccharides are assigned to the peaks as follows:N-acetyl galactosamine (GalNAc)at position 7.30; N-acetyl glucosamine(GlcNAc) at position 7.43; arabinose at position 10.52; rhamnose atposition 10.90; mannose at position 11.11; (note that the peak atposition 11.01 is a bubble; the mannose peak is just to the right ofthis peak); galactose at position 11.62; glucose at position 12.13.

[0061]FIG. 16: DEAE Sepharose Fast Flow of IBP-2. A sample was appliedon Pharmacia 1.0×30 cm column and eluted with piperazine/HCl (0.02 M, pH8.8) buffer at a rate of 5 mL/min. NaCl gradient was employed: 0-20% in20 column volumes, then 20-100% in 2 column volumes.

[0062]FIG. 17: DEAE Sepharose Fast Flow of retention portion after IBP-2was passed through an ultrafiltration membrane with MWCO lMDa. A samplewas applied on Pharmacia 1.0×30 cm column and eluted with piperazine/HCl(0.02 M, pH 8.8) buffer at a rate of 5 mL/min. NaCl gradient wasemployed: 0-20% in 20 column volumes, then 20-100% in 2 column volumes.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention relates to a method for enhancing immunitydefence mechanisms of mammals including humans. Immunity defences areenhanced by the administration of Chlorella-derived immunomodulators inthe form of Chlorella-derived extracts of molecular weights between1×10⁵ Da and 1×10⁷ Da.

[0064] A. Chlorella

[0065] Species of the Chlorella genus used in the invention includes thefollowing: minutissima, marina, salina, pyrenoidosa, vulgaris, anitrata,antarctica, autotrophica, regularis, among others (see World Catalog ofAlgae, 2^(nd) Edition, pages 58-74; Miyachi et al. (Eds); 1989; JapanScientific Societies Press, the content of which is herein incorporatedby reference).

[0066] Mutant strains of Chlorella, either naturally occurring orartificially produced, for example by irradiation (e.g. ultraviolet,X-ray), chemical mutagens or by site-directed mutagenesis, are withinthe scope of the invention. In one embodiment, C. pyrenoidosa and itsvariants are preferred. In another embodiment, C. ellipsoidea and itsvariants are preferred. Cultivation of Chlorella is carried out bymethods known in the art using suitable media and culture conditions(see for example, White and Barber, Biochimica Biophysica Acta., 1972,264:117-128). Polysaccharide production may be influenced byphysiological and metabolic manipulation. Composition of the growthmedia may influence growth rate leading to changes in cell wallthickness. Genes responsible for growth may be up- or down-regulated.For a method used to transform eukaryotic algae, see for example U.S.Pat. No. 6,027,900; for methods to select algal mutants, see for exampleU.S. Pat. No. 5,871,952. Thus, by selection under various conditions,variants of biopolymer immunomodulators from Chlorella may bemanufactured. B. Chlorella Extracts and Their Preparation

[0067] (a) Preparation of Crude Extract:

[0068] Crude aqueous extracts of Chlorella are prepared by methods knownin the art, including hot water extraction of cultured cells or spraydried cells (U.S. Pat. No. 4,831,020 and U.S. Pat. No. 5,780,096) andsolvent extraction methods (White and Barber, Biophys. Biochim. Acta,1972, 264:117-128; U.S. Pat. No. 3,462,412). Crude extracts may beobtained from the Taiwan Chlorella company(www.taiwanchlorella.com/product-3.htm).

[0069] In one embodiment, the crude extract is prepared from spray-driedChlorella cells with an average moisture content of 0.3%; (the moisturecontent was determined after drying the spray-dried material for 16 hunder a vacuum of 5 mm). The crude extract is prepared by treating thecells with aqueous media, preferably water or weak solutions of organicacids, such as acetic acid, ascorbic acid, benzoic acid, citric acid,lactic acid, maleic acid, propionioc acid, sorbic acid, succinic acidetc., preferably benzoic acid, under gentle agitation. The extractionprocess could be executed at various temperatures ranging from 0 to 100°C., preferably between 50 and 90° C. The yields and immunoactivitycorrelating with chromatographic profiles indicate that 1 h at 80° C. isa suitable combination of time and temperature to perform this stepefficiently.

[0070] The residual cells and the cell debris were separated bycentrifugation with a relative centrifugal force (RCF) of 150 to 10,000g, preferably 4,000 to 10,000 g. The time necessary to complete thisstep is in relation to the centrifugal force; 20 min. is sufficient at10,000 g. The supernatant was then micro-filtered. Alternatively,filtration may be used to remove whole cells and debris, in which caseuse of a series of filters starting from coarse, through medium andending with micro-filtration, is necessary. Cross-flow filtration orvibrating membrane technology is recommended to reduce fouling.Filtration is particularly sensitive to temperature and time periodrequired for extraction. Centrifugation is therefore the preferredroute.

[0071] After centrifugation or filtration, the supernatant (or filtrate)may be dried to obtain IBP-1 products in dry form. The drying wasachieved by lyophilization, cold airflow, or preferably by spray-drying.Alternatively, the volume of the extract could be reduced first (to10-50%, preferably 20%), and then the active materials precipitated fromthe solution with suitable precipitants, preferably ethanol or ammoniumsulfate.

[0072] IBP-1 was liberated from salts and low molecular mass products.Although a variety of aqueous media (such as diluted alcohols, variousbuffers, diluted acetic acid, etc.) could be employed, water was foundto be a sufficient medium for the dialysis. This step reduced the massof the extracted material by about 50% and increased its specificimmunoactivity (as judged by effect on stimulation of splenocytes, seebelow) by about 25%. Dialysis could be replaced by desalting with gelfiltration media, such as Sephadex G 25, Bio Gel P 6 or equivalents.Similarly, corresponding ultrafiltration membranes with correspondingmolecular weight cut-offs could be used. After the desalting step, thematerial (IBP-2) may be dried using methods described for IBP-1.

[0073] (b) Size Fractionation of Extracts:

[0074] Size fractionation of Chlorella extracts can be accomplished byany method known in the art, including size exclusion chromatography,sedimentation analysis e.g. gradient centrifugation, andultra-filtration.

[0075] IBP-1 or IBP-2 is a mixture of polysaccharides and polysaccharidecomplexes, with average molecular mass of the immunomodulatory fractionof interest ranging from 100 up to 10,000 KDa. Polysaccharide complexesare polysaccharides which are non-covalently associated with anon-polysaccharide biopolymer which, by itself, has no significantimmune activity. Non-polysaccharide biopolymers include DNA and proteinwhich may contribute to the cumulative molecular weight of the extractbut which has no significant immune activity.

[0076] In various embodiments, the high molecular weight polysaccharideand polysaccharide complexes are about 1×10⁵ Da to about 1×10⁵ Da, about3×10⁵ Da to about 5×10⁵ Da, about 5×10⁵ Da to about 6×10⁵ Da, about6×10⁵ Da to about 7×10⁵ Da, about 7×10⁵ Da to about 8×10⁵ Da, about8×10⁵ Da to about 9×10⁵ Da, about 9×10⁵ Da to about 1×10⁶ Da, about1×10⁶ Da to about 2×10⁶ Da, about 2×10⁶ Da to about 3×10⁶ Da, about3×10⁶ Da to about 4×10⁶ Da, about 4×10⁶ Da to about 5×10⁶ Da, about5×10⁶ Da to about 7×10⁶ Da, about 7×10⁶ Da to about 9×10⁶ Da, and about9×10⁶ Da to about 1×10⁷ Da.

[0077] Size fractionation to obtain the above fractions is based onprinciples of molecular sieving. Typically, size exclusionchromatography techniques and ultrafiltration methods are employed. Thebasic principles of size exclusion chromatography are well known tothose in the art, and are explained in “Gel filtration: Principles andMethods. Eighth edition., Amersham Pharmacia Biotech AB, Rahhms I Lund,Uppsala, Sweden”. The appropriate columns for fractionating particularranges can be readily selected and effectively used to resolve the abovefractions, e.g. Sephacryl S 100 HR, Sephacryl S 200 HR, Sephacryl S 300HR, Sephacryl S 400 HR and Sephacryl S 500 HR or their equivalents. Inan analogous fashion, Sepharose media or their equivalents, e.g.Sepharose 6B, 4B, 2B, could be used.

[0078] Purification of the polysaccharides or polysaccharide complexeswith protein could be achieved in combination with other chromatographytechniques, including affinity chromatography, ion exchange, hydrophobicinteraction chromatography etc. An example of IBP-2 (the retainate afterIBP-2 was passed through ultrafiltration membrane with MWCO>1 MDa)chromatography using DEAE-Sepharose Fast Flow anion exchangechromatography is given in FIGS. 16 and 17. The figures demmonstratesignificant decrease in the protein content after IBP-2 was passedthrough the ultrafiltration membrane.

[0079] Ultrafiltration of the samples could be performed using molecularmembranes with appropriate molecular mass cut-offs. The specificmembranes and procedures used to effect fractionation are widelyavailable to those skilled in the art, as outlined inhttp://www.uku.fi/laitokset/anat/PG/c_method.htm.

[0080] In one embodiment, the method used for characterising andquantifying these materials is based on combined size exclusionchromatography (SEC)/multi-angle laser light scattering(MALS)/refractive index detection (RI). In the hybrid technique(SEC/MALS/RI), an isocratic HPLC experiment using a Tosohaas GMPWXL SECcolumn is used to separate mixtures according to molecular size. On-lineMALS determines the average molecular weight distribution of the elutingbiopolymers and hence provides specificity in the analysis. RI detectionis used both for quantification and to provide the elution profilerequired in processing the MALS data.

[0081] An example of the SEC chromatogram obtained for a typical extractis shown in FIG. 1. The top trace is the chromatogram recorded using theRI detector while the bottom trace is the MALS response at one of thedetectors (90 degrees). The MALS peak is a maximum for the highmolecular mass component which actually corresponds to a smallpercentage of the total extract as can be seen from the upper trace.Thus, although the molecular weight range extends from a few KDa toabout 10 MDa, the weight average molecular weight (MW) for the entireextract is determined to be around 90 kDa. This can be seen most clearlyfrom the cumulative molecular weight profile of IBP-1 (FIG. 2).

[0082] IBP-1 or IBP-2 can be further fractionated using suitablechromatographic or ultrafiltration techniques. Size exclusionchromatography matrices with wide fractionation range such as SephacrylS 1000 SF (FIG. 3) resolved the extract into two peaks: the first elutedjust after the void volume; its average molecular mass, as measured byMALS, averaged 1,000 KDa; the second peak eluted just after the firstpeak. The combined fractions representing the first peak were desaltedand dried analogously to that of IBP-1, resulting in IBP-3. Thismaterial was superior in its immunoactivity compared to the second peak(IBP-4). Aqueous media were used in this chromatography procedure,preferably 0.15 M NaCl. Although IBP-1 exhibited higher immunoactivitythan the IBP-2, the difference was insignificant. The contribution tothe immunostimulant activity (increasing gradually with mass) startslevelling off when molecular mass reaches around 500 KDa.

[0083] IBP-4 could be further fractionated using suitablechromatographic or ultrafiltration techniques. Both ion exchangechromatography (IEC) and SEC were found to be useful for furtherresolution of IBP-4. SEC matrices with the appropriate fractionationrange, for example Sephacryl S 300, could be used. Sephacryl S 300 HRresolved IBP-1 or IBP-2 into two peaks (FIG. 4). The first peak startedeluting in the very last fractions of the void volume of the column. Themajority of the eluted biopolymers exhibited molecular masses rangingfrom 100 to 500 KDa. The combined fractions representing the first peakwere desalted and dried analogously to that of IBP-1, resulting inIBP-5. This material was superior in its immunoactivity to the secondpeak (IBP-6) which eluted just after the first peak touched the baseline(FIG. 5). IBP-5 represented typically only 30% of the combined mass ofboth peaks. Aqueous media were used for this chromatography procedure,preferably a 0.1M acetate, pH 4.5 buffer with linear NaCl gradient. Thedifference in the immunoactivity of IBP-5 and IBP-6 was higher than inthat between IBP-3 and IBP-4; typically the ratio of CPM_(IBP-3) toCPM_(IBP-4) was 5:2, as measured by the level of ³H-incorporation intoproliferating splenocytes. The process could be, to some extent,simplified by use of ultrafiltration membranes with molecular masscut-off of about 500 KDa. For instance, Omega ZM 500 membranesuccessfully could be used.

[0084] IBP-6 could be further fractionated using suitablechromatographic or ultrafiltration techniques, for instance usingchromatographic matrices such as Sephadex G 100, Sephadex G 75 oranalogous media or corresponding ultrafiltration membranes. However,specific immunoactivity residing in IBP-6 was significantly weaker thanthat of IBP-3 and IBP-5, and therefore was not of prime interest.However, Sephadex G 100 could be efficiently used to remove from IBP-2the majority of lower molecular weight material (IBP-8), which isassociated with low immunoactivity (FIG. 6). Typically the ratio ofCPM_(IBP-7) to CPM_(IBP-8) was 10:1; (IBP-7 being the high molecularmass and immunoactive fraction). This purification step could beachieved just as well with a YM-100 ultrafiltration membrane.

[0085] Crude extracts of Chlorella contain about 61% protein and 21%carbohydrate. Processing of Chlorella according to the present inventionresults in a higher percentage of polysaccharide and polysaccharidecomplexes, i.e. the extracts of the invention have a higher percentageof polysaccharide and polysaccharide complexes relative to the totalmaterial derived from Chlorella, compared to a crude extract from brokencells. It is understood that materials unrelated to Chlorella may beadded to the Chlorella extract and that such extracts are within thescope of the invention.

[0086] The percentage of polysaccharide and polysaccharide complexes inthe extracts of the invention is at least 23% (w/w) of the totalChlorella-derived content of the extract. In various embodiments, thepercentage is at least 24% (w/w), at least 26% (w/w), at least 28%(w/w), at least 30% (w/w), at least 35% (w/w), at least 40% (w/w), atleast 45% (w/w), at least 50% (w/w), or at least 60% (w/w).

[0087] The high molecular weight polysaccharide and polysaccharidecomplexes may be further purified and isolated to the variouspercentages indicated above by removal of non-polysaccharide components.Such non-polysaccharide components include DNA, RNA and unassociatedproteins. (Unassociated proteins are defined for the purpose of thepresent application as proteins which are not associated withpolysaccharides in a polysaccharide complex).

[0088] One method of removal is the use of digestion enzymes to cleavethe non-polysaccharide components, followed by size fractionation toremove the cleaved products as described in the Examples below (Example7). Digestion enzymes include pronase, ribonuclease, DNase andproteases, as well known in the art and described in various text books,one example of which is Maniatis et al., Molecular Cloning: A LaboratoryManual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. Proteases useful for digestion of unassociated proteins include:endo- and exopeptidases, pronase, serine proteases such as trypsin,chymotrypsin and subtilisin, thiol proteases such as papain, andcalcium-requiring proteases such as thermolysin.

[0089] Alternatively, non-polysaccharide components may be removed byaffinity chromatography, for example by use of DNA- or RNA-bindingmatrices (Maniatis et al., 1982). Another option is to purify thepolysaccharide and polysaccharide complexes away from the contaminatingcomponents by use of polysaccharide binding matrices such as lectins. Inanother embodiment, the extracts of the invention may be treated withglycosidic enzymes under conditions and for a length of time sufficientto effect cleavage of:

[0090] (i) three or more α-1,4-linked D-glucose units;

[0091] (ii) α-1,4-linked glucosides;

[0092] (iii) α-1,4-linked galactosides; or

[0093] (iv) α-1,4-linked D-glucose.

[0094] Examples of glycosidic enzymes useful for cleavage of suchglycosidic linkages include: amylase, amyloglucosidase, cellulase andneuraminidase.

[0095] C. Characterization of Extracts

[0096] Carbohydrate composition, DNA content and amino acid compositionof the Chlorella extracts of the invention can be determined by anysuitable method known in the art.

[0097] Immune activity of the extracts of the invention are associatedwith high molecular weight Chlorella polysaccharides, defined as thosemacromolecules consisting of monosaccharides joined by glycosidiclinkages. The polysaccharides are present in the extracts in the form offree polysaccharides or complexed polysaccharides (i.e. polysaccharideswhich are non-covalently associated with a non-polysaccharide biopolymerwhich, by itself, has no significant immune activity). In oneembodiment, the protein content of the extract is about 20% to 50%,preferably 20% to 30%. Of this percentage of proteins, about 40% to 60%are associated with polysaccharides.

[0098] Non-polysaccharide biopolymers include DNA, protein and possiblyRNA, which may contribute to the cumulative molecular weight of theextract but which has no significant immune activity. Unassociated RNA,DNA and protein, i.e. those not complexed with the polysaccharides, donot contribute significantly to immune activity of the extracts. For thepurposes of the present application, unassociated RNA, DNA and proteinare defined functionally as those RNA, DNA and protein which aresusceptible to cleavage by ribonuclease (RNAse), deoxyribonuclease(DNAse) and common proteases of the serine and thiol class. The extractsof the present invention may thus be essentially free or substantiallyfree of unassociated RNA, DNA and protein. By “essentially free” ismeant less than 5% unassociated DNA or RNA and less than 15%unassociated proteins. By “substantially free” is meant less than 2%associated DNA or RNA and less than 10% unassociated proteins.

[0099] It is understood that, while the non-polysaccharide biopolymersper se lack immune activity, their association with the polysaccharidesmay contribute to the immune activity of the polysaccharides since thenon-polysaccharide biopolymers of the complex may fulfill certain stericor polar requirements which enable the polysaccharides to functioneffectively as immunomodulators.

[0100] The extracts of the present invention may be digested withamylase, amyloglucosidase, cellulase and neuraminidase withoutsignificant loss of immune activity. Immune activity thus apparentlyresides in polysaccharides or their complexes which do not contain asubstantial amount of three or more α-1,4-linked D-glucose units;α-1,4-linked glucosides; α-1,4-linked galactosides; or α-1,4-linkedD-glucose. However, it is understood that immunomodulatorypolysaccharides may contain the above glycosidic linkages if suchlinkages are not accessible to enzyme digestion.

[0101] D. Uses of Extracts

[0102] Biological response modifiers have been defined as those agentsthat modify the host's biological response by a stimulation of theimmune system, which may result in various therapeutic effects. One ofthe categories of substances belonging to this class isimmunomodulators. As used herein, the term “immunomodulator” refers toan agent which is able to modulate an immune response. In the context ofthe present invention, such modulation is an enhancement of the host'simmunity defence mechanism.

[0103] Chlorella extracts are thought to be primarily a B-cell andmacrophage stimulator. One benefit of B-cell immunomodulators is thatthey can stimulate immune function in those who may have an impairedantibody response to an antigen. Also, a B-cell stimulator mightincrease the rapidity of the antibody immune response when presentedwith a new infection. Chlorella extracts provide a safe, efficacious andcost effective alternative for preventative health treatment.

[0104] In vitro studies demonstrated that Chlorella extracts stimulatedproliferation of BALB/c mouse spleen cells, and macrophage production ofIL-6 and NO₂. Chlorella extracts were also examined in viva, and foundto significantly reduce infection with Listeria monocytogenes, as wellas the fungus Candida albicans (see Examples 8 to 13).

[0105] A series of three toxicology trials have been completed forChlorella extracts. No effect of Chlorella extract administration wasevident during the 28-day oral toxicity study in rats. For the acuteoral toxicity in rats, to determine the highest non-lethal or the lowestlethal dose of the product following a single oral administration, thestudy found that the lowest lethal dose of a crude Chlorella extract wasin excess of 2000 mg/kg body weight. The bacterial mutation assay showedthat Chlorella extracts did not exhibit any mutagenic activity under thetest conditions.

[0106] A recently completed randomized, double-blind placebo-controlledstudy found that Chlorella extracts demonstrated significantimmunostimulatory effects in healthy adults receiving the influenzavaccine, compared to placebo subjects (see Example 14).

[0107] In vitro experiments with human blood cells show stimulation ofproduction of interleukins, similar to that seen in the mouse model.

[0108] Chlorella extracts of the invention are suitable for use in anycondition or disease state where immune response enhancement ormodulation is desired. In one embodiment, Chlorella extracts may be usedin an effective amount as adjuvants in various forms of mucosal vaccinepreparations, especially for oral administration.

[0109] Adjuvants may protect the antigen from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. Known adjuvantsfor mucosal administration include bacterial toxins, e.g., the choleratoxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT). Chlorella extracts,being an edible product of high molecular weight and themselves immunestimulants, are candidates for use as adjuvants in oral vaccines.

[0110] The term “effective amount” of an immunomodulator refers to anamount of an immunomodulator sufficient to enhance a host defencemechanism. This amount may vary to some degree depending on the mode ofadministration, but will be in the same general range. If more than oneimmunomodulator is used (for example, Chlorella extract in combinationwith echinacea), each one may be present in these amounts or the totalamount may fall within this range. The exact effective amount necessarycould vary from subject to subject, depending on the species, age andgeneral condition of the subject, the severity of the condition beingtreated, the mode of administration, etc. Thus, it is not possible tospecify an exact effective amount. However, the appropriate effectiveamount may be determined by one of ordinary skill in the art using onlyroutine experimentation or prior knowledge in the immunomodulator art.

[0111] The term “treatment” as used herein covers any treatment of amammal, particularly a human, and includes:

[0112] (i) preventing the disease from occurring in a subject which maybe predisposed to the disease but has not yet been diagnosed as havingit;

[0113] (ii) inhibiting the disease, i.e., arresting its development; or.

[0114] (iii) relieving the disease, i.e., causing regression of thedisease.

[0115] E. Form of Extracts

[0116] The nutritional and pharmaceutical compositions containingChlorella extracts of the invention may be formulated and administeredin any form suitable for enteral administration, for example oraladministration or tube feeding. The formulations are convenientlyadministered in the form of an aqueous liquid. The formulations suitablefor enteral application are accordingly preferably in aqueous form or inpowder or granulate form, including tablet form. The powder or granulatemay be conveniently added to water prior to use. In liquid form, thecompositions have a solid content of typically from 0.1% to 50% byweight, preferably from 1% to 10% by weight. As a drink, thecompositions may be obtained by any manner known, e.g. by admixing theChlorella extract with an energy source such as carbohydrates, fats andnitrogen sources.

[0117] The nutritional compositions may be in the form of a completeformula diet (in liquid or powder form), such that when used as solenutrition source, essentially all daily caloric, nitrogen, fatty acids,vitamin, mineral and trace element requirements are met. However, thenutritional compositions of the invention are preferably intended foruse as a dietary supplement.

[0118] Pharmaceutical compositions of the invention may also beformulated in a single-dose format, where they comprise Chlorellaextracts and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions are suitable for enteral administration, such as oral,nasal or rectal administration. Suitable compositions may be in liquidform or solid form. Dosage of liquid compositions are typically from0.1% to 50% by weight, preferably from 1% to 10% by weight of Chlorellaextract. Dosage of solid compositions are typically from 0.2 mg/kg to200 mg/kg, preferably from 1 mg/kg to 10 mg/kg of Chlorella extract Thecompositions may be in the form of tablets, hard and soft capsules, andsachets.

[0119] Suitable carriers are known in the art. They comprise fillerssuch as sugars or cellulose, binders such as starch, and disintegratorsif required.

[0120] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1 Preparation of IBP-1

[0121] Twenty grams of dried powder of Chlorella pyreinodosa were mixedwith 100 mL distilled water and heated under gentle stirring for 1 h at80° C. The material was centrifuged at 10,000 g for 20 min; the residualpellet was washed twice. The combined supernatants were thenmicro-filtered and spray dried. The average yield was 10.5%. Theextraction of the pellet for 1 h at 80° C. could be repeated severaltimes, preferably twice, instead of simple wash.

[0122] Alternatively, the combined supernatants were evaporated to ⅕ oftheir original volume using a rotary evaporator under reduced pressure,then precipitated with ethanol (80% final ethanol content). The mixturewas kept at 4° C. for 18 h, then the precipitate was filtered off,washed with absolute ethanol and vacuum-dried.

EXAMPLE 2 Preparation of IBP-2

[0123] The extracted material, prepared as above, was desalted byexhaustive dialysis against water at 4° C. and then spray-dried. Theaverage yield was about 7.5%.

EXAMPLE 3 Chromatography of IBP-2 and Preparation of IBP-3

[0124] Twenty milligrams of IBP-2 were dissolved in 1 mL of distilledwater, pre-filtered with a 0.45 μm filter and loaded on Sephacryl S 1000HR column (1.0×50 cm) and eluted with 0.15 M NaCl. Chromatography wasmonitored at 280 nm and at 490 nm after interaction of the elutedfractions with a phenol/sulfuric acid reagent. The mixture was resolvedinto two peaks. The major immunoactivity was in the high molecular masspeak (IBP-3). However, the lower molecular mass peak (IBP-4) alsocontained a significant portion of immunoactivity. The chromatogram isshown in FIG. 3.

EXAMPLE 4 Chromatography of IBP-2 and Preparation of IBP-5

[0125] Two hundred mg of IBP-2 were dissolved in 10 mL distilled water,pre-filtered with 0.45 μm filter, loaded on a Sephacryl S 300 HR column(2.5×90 cm), and eluted with 0.1 M acetate buffer, pH 4.5 and linearNaCl gradient. Chromatography was monitored at 280 nm, and at 490 nmafter interaction of the eluted fractions with-a phenol/sulfuric acidreagent. The mixture was resolved into two peaks. The majorimmunoactivity was in the high molecular mass peak (IBP-5). The lowermolecular mass peak (IBP-6) consisted of two fused peaks (an apparentpeak shoulder, the fractions of which were tested as IBP-6s, FIG. 5).The SEC profile is shown in FIG. 4.

EXAMPLE 5 Chromatography of IBP-2 and Preparation of IBP-7

[0126] Two hundred mg of IBP-2 were dissolved in 10 mL distilled water,pre-filtered with 0.45 μm filter, loaded on Sephadex G 100 column(2.5×90 cm), and eluted with 0.1 M acetate buffer, pH 4.5 and linearNaCl gradient. Chromatography was monitored at 280 nm, and at 490 nmafter interaction of the eluted fractions with a phenol/sulfuric acidreagent. The mixture was resolved into two peaks. The majorimmunoactivity was in the high molecular mass peak (IBP-7). The lowermolecular mass peak (IBP-8) retained much less of the activity. IBP-9represents the fractions following IBP-8 (FIG. 6).

EXAMPLE 6 Compositional Analysis

[0127] Dialysis or ultrafiltration demonstrated that only a smallportion of immunoactivity was associated with low molecular mass (lessthan 100 KDa) compounds. The active material is thermally stable andcould be precipitated from solution with ethanol or ammonium sulfate.The thermal stability of the extract makes it suitable for spray-drying;indeed, immunomodulatory activity was retained after the spray-dryingprocess. A typical protein content of a fraction is about 30%. DNAcontent varies from 0% to 20%, with 0% to 2% at molecular masses greaterthan 100 KDa.

[0128] (a) Carbohydrate Composition by Planar Chromatography and GasChromatograph (GC):

[0129] In one embodiment, the lyophilized extract (or further purifiedfractions) was dissolved in water (1 mg/mL, 400 μL) and hydrolysed with1M trifluoacetic acid (TFA, 1 mL) at 100° C., overnight with stirring,in tightly sealed 4 mL screw cap vials. The sample was then evaporatedrepeatedly to dryness using methanol. The dried hydrolysate was reducedusing 0.5 M NaBH₄ in 1 M NH₄OH (0.6 mL) under overnight stirring at roomtemperature. The borohydride was then quenched with acidic methanol (20%acetic acid in methanol, 1 mL) and the mixture evaporated to dryness.

[0130] Three mL of acetic anhydride were added to the sample; themixture was heated in a water bath to 80° C. for 2 h to produce acetylalditols and then evaporated to dryness.

[0131] The acetyl alditol samples were extracted by distributing thereaction mixture between ethyl acetate and water; the organic phase wasused directly for gas chromatography/mass spectroscopy (GC-MS) analysis.

[0132] All standards and sample extracts were dissolved in ethyl acetateand concentrated to approximately 100 μL. Samples were injected usingthe split mode of a Thermoquest Trace 2000 gas chromatograph at a 10:1split and chromatographed on an SGE BPX70 capillary column (30 m×0.25mm×0.25 μm film thickness). Helium carrier gas was used at a constantflow rate of 1.0 mL/min.

[0133] The gas chromatograph oven was programmed at an initialtemperature of 190° C. (hold for 1 minute) followed by a 3° C./minuteramp to 260° C. (hold for 10 minutes at 260° C.). The capillary columnwas interfaced directly to the mass spectrometer (Thermoquest GCQ iontrap), with the transfer line temperature at 260° C. Using this ovenprogram, all compounds of interest were found to elute within 20minutes.

[0134] The mass spectrometer ion source was maintained at 150° C.Spectra were recorded from m/z 50 to 500 using both electron impact mode(70 eV) and chemical ionization (CI) mode with ammonia reagent gas.

[0135] Retention times for the monosaccharides were established byderivatizing pure standards of individual sugars and/or chromatographingcommercially available mixtures of alditol acetates (Supelco, Inc.). Thesugars present in the sample extracts were identified by comparison ofretention times and mass spectra against these standards. All sampleswith immunomodulatory activity contain glucose, galactose, rhamnose,mannose and arabinose. Extracts of molecular weight greater than 1×10⁶Da are substantially free of ribose.

[0136] (b) Monosaccharide Compositional Analysis Using PolyacrylamideGel Electrophoresis (PAGE):

[0137] The standard hydrolysis protocols to release acidic, neutral orbasic monosaccharides from the polysaccharide backbones were employed.The liberated monosaccharides were labeled with fluorescent labelingreagent 2-aminoacridone (AMAC) first, followed by reduction of theformed Schiff base with sodium cyanoborohydride. Polyacrylamide gelelectrophoresis of the mixtures was then run on gradient gels accordingthe instruction manual. Hydrolysates produced from IBP-2 containedglucose, galactose, rhamnose mannose, and arabinose. A substantial mountof ribose was also found. However, the hydrolysates produced bytreatment of IBP-5 and IBP-7 with 2M TFA at 100° C. for 5 h clearlycontained only glucose and galactose as well as rhamnose, mannose andarabinose. No ribose was found in these hydrolysates. This was in accordwith our previous findings that RNA fragments of low molecular mass werepresent in IBP-2. However, because of their small size they could not bepresent in high molecular mass peaks of IBP-5 and IBP-7 obtained fromSephadex G 100 of Sephacryl S 300 chromatographies, respectively.Another band placed between N-acetyl galactosamine (GalNAc) and mannosewas detected in PAGE but could not be assigned to any of theconventional monosaccharides. The reaction mixtures obtained byhydrolysis performed under the conditions used for sialic acid release(0.1 TFA, 80° C., 1 h) resulted in a product with a Rf identical withthat of sialic acid and another major band with retention timesignificantly slower than that corresponding to GalNAc. Four molar HClhydrolysis for 3 h at 100° C. (a condition for aminosugar release)resulted in PAGE in bands corresponding to GalNAc and N-acetylglucosamine (GlcNAc). However judging from the intensity of other bandsthey were insignificant components of IBP-5 of IBP-7.

[0138] (c) Monosaccharide Compositional Analysis Using CapillaryElectrophoresis (CE):

[0139] The protocol followed was that of Sato et al. (Sato K., Okubo A.,Yamazaki T., (1997) Determination of monosaccharide derivatized with2-aminobenzoic acid by capillary electrophoresis. Anal Biochem 251:119-121). IBP-2 or its fractions were hydrolyzed first in 0.1 M TFA (1mg/ml), at 100° C. for 18 h, the aqueous acid was removed under reducedpressure, the residual TFA was removed by a sequential evaporation withmethanol until dryness.

[0140] The crude extract IBP-2 contains primarily glucose and galactose;glucose is the most prevalent monosaccharide in the extract. Alsopresent are rhamnose, mannose and arabinose, albeit in significantlysmaller quantities (FIG. 14). When the extract was subjected toultrafiltration using a 1 Mda MWCO membrane (FIG. 15), the ratio betweenglucose and galactose changed and the ribose peak disappeared,indicating (in agreement with PAGE data) removal of RNA and itsfragments.

[0141] (d) Amino Acid Composition:

[0142] The free amino acids contained in the extracts were determined asfollows: The samples were dissolved in distilled de-ionized water for afinal concentration of 10 mg/mL. To 25 μL (250 μg) of each sample, 75 μLof Beckman sample buffer (Na-S) was added. Samples were centrifuged at16,000×g to remove particulate matter before analysis on the BeckmanModel 6300 amino acid analyser. The centrifugation clarified thesolution and the pellet was retained; the supernatant was analysed forfree amino acids.

[0143] To determine the amino acid composition of whole hydrolysates,portions of each sample (250 μg) were placed into a cleaned glass testtube along with the internal standard norleucine and 1 mL 6N HCl. Thetubes were sealed under vacuum and the peptides and polypeptideshydrolysed at 105° C. for 20 hours. The tubes were opened and thesamples were dried in a Savant Environmental Speed-vac at roomtemperature. The residues were re-dissolved in Na-S buffer and handledas described above for the determination of free amino acids.

[0144] (e) DNA Content:

[0145] The Chlorella extract was dissolved in distilled water (500 μL).An equal volume of a phenol/chloroform/isoamyl alcohol (25:24:1, v/v)solution is added. The mixture is shaken vigorously then centrifuged at13,000 rpm for 5 minutes. The top aqueous layer is pipetted off andre-extracted with an additional 500 μL of the organic mixture. Theaqueous layer is re-extracted until the protein layer (a visibleinterface between the aqueous and organic layers) is negligible. Afterthe final protein extraction, the aqueous layer was transferred to afresh tube and to it was added glycogen (10 μL), 3M sodium acetate (50μL), and cold ethanol (1 mL). The mixture was shaken and placed in the−80° C. freezer for an hour. It was then centrifuged at 13,000 rpm for15 minutes and the supernatant was poured off. The pellet was dried onthe speed-vacuuming and re-dissolved in water. Its absorbance wasmeasured at 260 nm against a water blank. Since proteins also absorb at260 nm, protein absorbance readings at 280 nm were also taken as ameasure of protein content in the DNA sample.

EXAMPLE 7 Enzyme Digestion of Extracts

[0146] Enzyme degradation was used as a technique to selectivelyeliminate various macromolecular classes from the Chlorella extracts.IBP-2 or its fractions (both IBP-2 and its fractions are designatedONC-107 in this example) were treated with pronase, DNAse, RNAse,amylase, amyloglucosidase, cellulase and neuraminidase in separateexperiments. SDS page and agarose electrophoresis as well as thin layerchromatography were used to monitor the enzymatic reactions (seesections (a) to (g) below). The final reaction mixtures were dialyzed,lyophilized and tested for the capacity to stimulate undifferentiatedspleen cells by thymidine incorporation into murine splenocytes(Examples 9 and 11). The results are summarized as follows:

[0147] No effect on the capacity to stimulate undifferentiated spleencells within a statistical significance after treatment with theselected enzymes.

[0148] No presence of large molecular mass RNA in IBP-2. This is inagreement with other findings (above), specifically that no ribose wasfound in high molecular mass species (IBP-5, IBP-7) and in fractionsultrafiltered with MWCO>1 MDa. This clearly demonstrates that RNA is notthe primary source of the immunostimulating activity.

[0149] A very small quantity of DNA was found, which is in accord withour data obtained from the isolation of DNA using a silicon carbidecolumn (about 2% content, 500 bp) (see Haj-Ahmad Y (1999) Nucleic acidpurification and process. Canadian published application 2,270,106).

[0150] All enzyme digestions were performed in parallel with positivecontrols to ensure that the enzymes were active. The experiments clearlyindicate that unassociated, enzyme-accessible protein and nucleic acidsare unlikely to be sources of the activity. The source of immunoactivityis a polysaccharide (perhaps complexed with another biopolymer which hasno significant effect by itself and which might have an indirect role,e.g. stability). The immunoactivity of the polysaccharide is notaffected by cleavages in the regions of three or more α-1,4-linkedD-glucose units, α-1,4-linked glucosides and galactosides, orα-1,4-linked D-glucose, assuming such linkages are accessible to enzymecleavage.

[0151] (a) Protease Treatment

[0152] Protease (100 μg/ml, Streptomyces griseus) was added to ONC-107(20 mg/ml) in TRIS buffer (0.05M, pH 7.4) and incubated for 1 hour at36° C. Incubation was stopped by heat deactivation of the enzyme at 80°C. for 1 hour followed by centrifugation for 10 minutes at 13000 rpm.Aliquots were taken from the solution at time intervals of 0, 5, 10, 15,30 and 60 minutes and prepared for analysis by SDS-PAGE electrophoresis(12% gel, total protein stain). The optimum concentration of proteasewas determined by a similar electrophoretic time course involving BSA (1mg/ml). The final digest mixture was analyzed by agarose electrophoresis(1% stained with ethidium bromide).

[0153] (b) DNAse Treatment

[0154] DNAse (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer(0.0SM, pH 7.4, 10 mM MgCl₂) and incubated for 1 hour at 36° C.Incubation was stopped by heat deactivation of the enzyme at 80° C. for1 hour followed by centrifugation for 10 minutes at 13000 rpm. The finaldigest mixture was analyzed by SDS-PAGE electrophoresis (12% gel, totalprotein stain) to check for protein degradation. Agarose electrophoresis(1% stained with ethidium bromide) was used to confirm nucleic aciddegradation.

[0155] (c) RNAse Treatment

[0156] RNAse (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer(0.05M, pH 7.4, 10 mM NaCl) and incubated for 1 hour at 36° C.Incubation was stopped by heat deactivation of the enzyme at 80° C. for1 hour followed by centrifugation for 10 minutes at 13000 rpm. The finaldigest mixture was analyzed by SDS-PAGE electrophoresis (12% gel, totalprotein stain) to check for protein degradation. Agarose electrophoresis(1% stained with ethidium bromide) was used to confirm nucleic aciddegradation.

[0157] (d) Amylase Treatment

[0158] Amylase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRISbuffer (0.05M, pH 7.4) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. The final digest mixturewas analyzed by SDS-PAGE electrophoresis (12% gel, total protein stain)to check for protein degradation. Agarose electrophoresis (1% stainedwith ethidium bromide) was used to confirm nucleic acid degradation.

[0159] Additionally, thin layer chromatography (TLC) confirmed theactivity of the enzyme as follows. Amylase (100 ug/ml) was added to asolution of starch (1 mg/ml) and incubated for one hour at 36° C. Thesolution was analyzed via TLC using keisgel silica plates eluted withisopropanol:ethylacetate:water (7:1:2). The plates were developed after10 minutes drying in the horizontal position using a sulfuricacid/ethanol solution. Glucose (1 mg/ml) was used as a control as wasuntreated starch. Treatment of starch with amylase resulted in theliberation of glucose.

[0160] (e) Amyloglucosidase

[0161] Amyloglucosidase (100 μg/ml) was added to ONC-107 (20 mg/ml) inTRIS buffer (0.05M, pH 4.4) and incubated for 1 hour at 36° C.Incubation was stopped by heat deactivation of the enzyme at 80° C. for1 hour followed by centrifugation for 10 minutes at 13000 rpm. TLCconfirmed the activity of the enzyme as follows. Amyloglucosidase (100ug/ml) was added to a solution of starch (1 mg/ml) and incubated for onehour at 36° C. The solution was analyzed via TLC using keisgel silicaplates eluted with isopropanol:ethylacetate:water (7:1:2). The plateswere developed after 10 minutes drying in the horizontal position usinga sulfuric acid/ethanol solution. Glucose (1 mg/ml) was used as acontrol as was untreated starch. Treatment of starch with theamyloglucosidase resulted in the liberation of glucose.

[0162] (f) Cellulase

[0163] Cellulase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRISbuffer (0.05M, pH 7.4) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. TLC confirmed theactivity of the enzyme as follows. Cellulase (100 ug/ml) was added to asolution of cellulose (1 mg/ml) and incubated for one hour at 36° C. Thesolution was analyzed via TLC using keisgel silica plates eluted withisopropanol:ethylacetate:water (7:1:2). The plates were developed after10 minutes drying in the horizontal position using a sulfuricacid/ethanol solution. Glucose (1 mg/ml) was used as a control as wasuntreated cellulose. Treatment of cellulose with cellulase resulted inthe liberation of glucose.

[0164] (g) Neurominidase

[0165] Neurominidase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRISbuffer (0.05M, pH 5.0) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. TLC confirmed theactivity of the enzyme as follows. Neurominidase (100 ug/ml) was addedto a solution of N-acetyl-neuromidose (1 mg/ml) and incubated for onehour at 36° C. The solution was analyzed via TLC using keisgel silicaplates eluted with isopropanol:ethylacetate:water (7:1:2). The plateswere developed after 10 minutes drying in the horizontal position usinga sulfuric acid/ethanol solution.

EXAMPLE 8 Stimulation of Splenocyte Proliferation

[0166] Fresh splenocyte cells (splenocyte primary culture) were platedat 3×10⁵ cells/well in cRPMI medium in a 100 μl volume in 96-well flatbottom tissue culture plates. Test samples in 100 μl of cell medium wereadded to triplicate wells giving a final total volume in each well of200 μl. The plates were covered with sterile lids and incubated in 5%CO₂ at 37° C. and 100% humidity in a CO₂ incubator for 48 h. The cellswere then pulsed with ³H-thymidine (1 μCi per well in 10 μl cRPMI) andincubated for 18 h. Cells were harvested with an automated cellharvester equipped with filter strips. The filter strips were allowed todry for 3 h at 37° C. The radioactivity incorporated by the cells wasdetermined by counting the filter strips immersed in scintillationmedium in a liquid scintillation counter. An example of the experimentis illustrated in FIG. 7. A comparison of applicant's extract with twocommercial samples is illustrated in FIG. 13.

EXAMPLE 9 Isolation of B-Cells and T-Cell from Splenocytes andStimulation of the Cells

[0167] Mouse splenocytes were isolated by routine methods and placed intissue culture flasks at 37° C. for 2 hr, to allow the macrophages tosettle and adhere to the flask. The lymphocytes (which are suspended anddo not adhere) are then removed and placed in a 50 ml centrifuge tube,spun and resuspended in a small amount of cold PBS-EDTA-BSA buffer (pH7.4; 1-3 ml) and placed on ice. Cells were counted and recentrifuged.They were then resuspended at 1×10⁸ in 0.3 ml of the PBS-EDTA-BSA bufferin a 15 ml centrifuge tube.

[0168] Negative isolation of B-cells: 100 μl of Miltenyi microbeadscoated with anti-Thy-1.2 antibody was added to the resuspended cells,and the mixture was incubated for 20 minutes. PBS-EDTA-BSA (5 mL) wasadded and the suspension was pipetted into a midiMACS column in amidiMACS magnet. The column had a 25-gauge needle on the outflow torestrict the flow rate. The T-cells, bound to the anti-Thy-1.2antibody-coated magnetic beads, adhered to the column. The B-cellspassed through and were collected. The column, without being removedfrom the magnet, was rinsed with 5 ml of the buffer to remove anyresidual B-cells. The B-cells were combined, spun, resuspended in 1 mlof cRPMI and counted. They were then resuspended to 5×10⁶ cells per mlof cRPMI and plated at 100 μl/well for stimulation assays.

[0169] Negative isolation of T-cells: The same procedure was followed asfor isolation of B-cells, except the magnetic beads used for theincubation were coated with anti-B220 antibody instead of anti-Thy-1.2.

[0170] The purified T- and B-cell populations were tested as describedin Example 8. The results are shown in FIG. 8.

EXAMPLE 10 Nitrite Assay for Mouse Peritoneal Macrophages

[0171] Mice were euthanized by cervical dislocation, and placedspreadeagled on their backs. Their abdomens were sterilized with 70%alcohol, and a careful midline incision exposing the INTACT peritonealwall was made. A 10-ml syringe was used to inject 10 ml of coldcRPMI-1640 into the mouse peritoneal cavity. The mice were gently rockedfrom side to side with the needle still inserted. The cRPMI-1640containing the peritoneal macrophages was slowly drawn from theperitoneal cavity through the needle. About 8 ml of fluid per mouse wasrecovered. The peritoneal fluid was pooled and put into 50 mL centrifugetubes on ice. The cells were spun down, washed and resuspended in 1 mlof cRPMI-1640. After counting, they were resuspended to 1-2×10⁶ cells/mland plated into a 96-well tissue culture plate at 1×10⁵ cells/well in a100 μl volume. Test samples were added in cRPMI (100 μl) with andwithout IFN-μ. The positive controls were the cells +IFN-μ andLPS+IFN-μ. The cells were incubated for 48 h in a CO₂ incubator in 5%CO₂.

[0172] Fifty μl of the culture fluid was collected and transferred towells in a 96-well flat bottom ELISA plate. Twofold serial dilutions ofNaNO₂ (125 μM to 1 pM final concentration) in cRPMI were made, and 50 μlof each Greiss Reagent solution and the NaNO₂ dilutions were added to aset of wells to provide a standard curve. Absorbance at 550 nm wasmeasured and a plot of absorbance values against NaNO₂ concentrationswas made. The standard curve was used to determine the amount of NO₂produced by the peritoneal macrophage samples. An example of theexperiment is illustrated in FIG. 9.

EXAMPLE 11 Determination by Sandwich ELISA of Stimulation of CytokineProduction by Mouse Splenocytes

[0173] Cytokine production was measured in mouse splenocytes, inseparated mouse T- and B-lymphocytes, and in mouse macrophages. The testsamples were added at several concentrations to the cells in microtitertissue culture plates. After incubating for 24-48 h, depending on thecytokine of interest, the supernatant culture fluid was removed forELISA.

[0174] ELISA plates were coated with anti-cytokine monoclonal antibodiesby incubation at 4° C. overnight in a carbonate buffer, pH 9.6. Theplates were then washed with Tris-buffered saline (TBS), post-coatedwith 2 mg/ml BSA in TBS, (200 μl/well) for 2 h at room temperature andwashed with TBS/Tween. The samples and standards (the latter diluted 1ng/ml to 15 pg/ml in twofold dilutions) were diluted in TBS/Tweencontaining 1 mg/ml BSA (100 μl/well), added to the plate, incubatedovernight at 4° C. and then washed with TBS-Tween.

[0175] The appropriate biotinylated anti-cytokine mAb (0.5 μg/ml) inPBS-Tween containing 1 mg/ml BSA (100 μl/well), was added. The plate wasincubated at room temperature for 1 h and then washed with TBS-Tween.Extravidin-Peroxidase in PBS-Tween containing 1 mg/ml BSA (100 μl/well)was added and incubated at room temperature for 30 minutes. The plateswere then washed. One hundred μl/well of TMB substrate solution wasadded, and after 10 to 30 minutes, depending on color development, thereaction was stopped with 100 μl/well of 1 M H₃PO₄. The plate was readat 450 nm. An example of stimulation of IL-6 production is illustratedin FIG. 10.

EXAMPLE 12 Effect of IBP-2 on Proliferation of Listeria monocytogenes inInfected Mice

[0176] Two doses of IBP-2 (0.1 mg or 4 mg) or plain water (negativecontrol) were administered to Balb/c mice by intragastric tube threetimes a week for four weeks. The mice were then infected by intravenousinjection of 5,000 viable Listeria monocytogenes organisms. Three daysafter the Listeria injections, the mice were sacrificed. Cellsuspensions of their spleens were made and cultured on culture dishes todetermine the number of bacteria in the spleens. The water-fed animalshad 92,202 (±23,000) bacterial colony forming units (CFUs) in theirspleens; (the number in bracket refers to the standard deviation). Theanimals fed 0.1 mg of IBP-2 per dose had 43,310 (±7,021) CFUs and theanimals fed 4 mg of IBP-2 per dose had only 5,317 (±492) CFUs (p<0.05)(FIG. 11).

EXAMPLE 13 Effect of IBP-2 on Proliferation of Candida albicans inInfected Mice

[0177] IBP-2 (4 mg), crude algae (4 mg or 20 mg), or plain water wasadministered to Balb/c mice by intragastric tube 3 times per week fortwo weeks. The mice were then infected by intravenous injection of500,000 viable Candida albicans organisms. Feeding continued until themice were sacrificed 12 days after infection. The kidneys were removedand cell suspensions made and cultured on Sabouraud agar to determinethe number of C. albicans colonies which developed overnight. Thefollowing results were obtained: water fed mice 594 (±556) colonies(mean±standard deviation); IBP-2 (4 mg) fed mice 42 (±75) (p<0.05compared to water fed mice); algae (4 mg) fed mice 335 (±663); algae (20mg) fed mice 79 (±70); (statistically, the p value for this groupcompared to the water fed group was >0.05, although it was very close tobeing significant). The results are illustrated in FIG. 12.

EXAMPLE 14 Phase 2 Clinical Trial Study

[0178] This study was designed to assess the immunostimulatory efficacyof Chlorella extracts as a nutritional supplement in healthy adults over50 years of age. A first-in-man study was completed which demonstratedthe safety and tolerability of a Chlorella extract corresponding toIBP-1 when given as a once daily supplement over three weeks. Thispurpose of this phase 2 study was to increase the human experience withChlorella extracts through increased safety and tolerability assessmentand to explore the capacity of Chlorella extracts as an immunestimulating nutritional supplement in humans.

[0179] The study was designed as a randomized, placebo controlledclinical trial in which adults 50 years of age or older were assigned bychance to receive a 200 mg capsule of a Chlorella extract correspondingto IBP-1, (designated ONC-107 for the purpose of this study), a 400 mgcapsule of ONC-107, or a placebo capsule (containing no ONC-107). Whilethe trial was underway, the investigators, the nurses and other studypersonnel and the participants were not aware to which group they wereassigned. The safety and side effects were measured by careful recordingby the participant of any adverse event and reporting these to studypersonnel. Specific adverse events measured included fever, abdominalpain, nausea, vomiting, diarrhea, fatigue, decreased appetite, headache,body aches, sore joints, and rash. Safety was also measured by a seriesof blood tests before starting the study capsules and at the completionof the study capsules. These tests included tests of liver function(AST, ALT), blood profiles (complete blood count), and immunologicalfunction (ANA, anti-DNA, rheumatoid factor, Coombs, C3, C4, quantitativeIgG, IgA, IgM, and IgE). Medication was taken for 28 days; after 21days, participants were immunized with a commercially available,inactivated, split virion influenza vaccine. Antibody response to theimmunization was assessed by measuring antibodies to the three influenzavirus strains before, and 7 and 21 days after immunization. Cellmediated immunity was measured by evaluating the response to aninfluenza skin test at the beginning of the study and one week after theimmunization.

[0180] A total of 124 subjects were enrolled into the study and took thestudy medication; 7 participants withdrew from the study. Only oneparticipant withdrew because of adverse events (nausea and abdominalpain). The three treatment groups were similar in age, gender andbaseline history and physical examination at the commencement of thestudy. The majority of participants were women (73.2-80.5% of eachgroup). Participant compliance was excellent. Antibody response to theinfluenza vaccine was not significantly higher in the ONC-107 treatedparticipants overall although in participants 55 years of age or youngerthere was a significantly enhanced response to some antibody measures(and a consistent trend to the others). There were no serious adverseevents in any of the study participants. An adverse event was reportedby most participants at sometime during the study but, for the mostpart, these events were not reported more frequently in the ONC-107recipients compared to the placebo recipients (fatigue was reported morefrequently by 200 mg ONC-107 recipients and abdominal pain morefrequently by placebo recipients older than 55 years of age). There wereno significant changes in laboratory measurements before and aftertherapy, or between recipients of the ONC-107 and placebo.

[0181] The results of this phase 2 study of the nutritional supplementONC-107 at doses of 200 mg and 400 mg for 28 days in healthy adults areoutlined below. They indicate that, despite an age effect, this productis well tolerated and safe for oral administration and has a measurableimmunostimulatory effect.

[0182] The immunostimulatory effect was measured by antibody response toinfluenza vaccine in healthy adults over 50 years of age, although theseresponses were in general limited to the younger cohort in the studypopulation (50-55 years of age). The increase in antibody response inthis subgroup was statistically significant for some of the comparisonsbut the trend was apparent in all serological comparisons made. Thepre-study hypothesis that the effect of ONC-107 would be best observedin the older subjects because of their decreased responsiveness toinfluenza vaccine was not supported by the data; in contrast, it was theyounger, more responsive subjects who demonstrated an effect of theONC-107.

[0183] The younger subjects tended to show immunostimulatory effects ofONC-107 (especially 400 mg dose). A potential reason for the lack ofeffect in the older age group was that the older group may have hadhigher pre-immunization antibody titers which may indicate a greaterdegree of prior exposure to antigenically similar flu virus A strains(but not the B strains).

[0184] 1. For the A/Caledonia strain of the flu vaccine, the younger(<=55 yrs.) age group had higher mean antibody titers with both dosesthan placebo at both 7 (not significant at 7 d) and 21 days (p=0.05):placebo=43.2; 200 mg=84.3; 400 mg=84.4.

[0185] 2. For the B/Yamanashi strain of the flu vaccine, the 400 mggroup had higher titers (30.1) at 7 days vs. placebo (14.4, p=0.03), butthis was not significant at 21 days. The 200 mg dose was notsignificantly greater than placebo at 7 days (15.9) or 21 days (25.3).

[0186] 3. For A/Panama strain of the flu vaccine, there was a similartrend in favour of 400 mg dose, but it was not significant. At 7 days,the 400 mg group had titers of 64.5, compared to placebo (39.9), whichis not significant. The 200 mg group has titers of 26.6. At 21 days,there was also a trend in favour of 400 mg vs. placebo; again, 200 mgwas not better than placebo (57.4).

[0187] Similar trends were observed for the proportion of subjectshaving 2- and 4-fold antibody responses.

[0188] For the B/Yamanashi in the <55 yr. group, only 5% of the placebohad a 2-fold antibody response at 7 days. This is in contrast with the400 mg group (41.2%, p=0.01) and 200 mg group (6.3%, p=0.04).

[0189] There was also a non-significant trend for an increased responseat 21 days, and a non-significant trend in the proportion of the <55 yr.age group achieving seroprotective levels of 40 RD (reciprocaldilutions) at 7 days post-immunization.

[0190] Numerous modifications, variations and adaptations may be made tothe particular embodiments of the invention described above withoutdeparting from the scope of the invention which is defined in theclaims.

1. An extract comprising high molecular weight Chlorella polysaccharideand polysaccharide complexes, wherein the high molecular weightpolysaccharide and polysaccharide complexes are about 1×10⁵ Da to about1×10⁷ Da and constitute at least 22% (w/w) of the totalChlorella-derived content of the extract.
 2. The extract of claim 1wherein the high molecular weight polysaccharide and polysaccharidecomplexes are about 5×10⁵ Da to about 5×10⁶ Da.
 3. The extract of claim1 wherein the high molecular weight polysaccharide and polysaccharidecomplexes constitute at least 26% (w/w) of the total Chlorella-derivedcontent of the extract.
 4. The extract of claim 1 wherein the highmolecular weight polysaccharide and polysaccharide complexes constituteat least 30% (w/w) of the total Chlorella-derived content of theextract.
 5. The extract of claim 1 wherein the high molecular weightpolysaccharide and polysaccharide complexes contain glucose and at leastone monosaccharide selected from the group consisting of: galactose,rhamnose, mannose and arabinose.
 6. The extract of claim 5 wherein thehigh molecular weight polysaccharide and polysaccharide complexes aresubstantially free of ribose.
 7. The extract of claim 1 which issubstantially free of nucleic acids and ribonucleic acids.
 8. Theextract of claim 5 wherein the high molecular weight polysaccharide andpolysaccharide complexes also contain N-acetyl glucosamine and N-acetylgalactosamine.
 9. The extract of claim 1 which is substantially free ofunassociated proteins.
 10. An extract consisting of high molecularweight Chlorella polysaccharide and polysaccharide complexes, whereinthe high molecular weight polysaccharide and polysaccharide complexesare about 1×10⁵ Da to about 1×10⁷ Da.
 11. The extract of claim 10wherein the high molecular weight polysaccharide and polysaccharidecomplexes are about 1×10⁶ Da to about 5×10⁶ Da.
 12. The extract of claim1 which retains immunomodulating activity upon treatment underconditions and for a length of time sufficient to effect digestion ofunassociated DNA.
 13. The extract of claim 1 which retainsimmunomodulating activity upon treatment under conditions and for alength of time sufficient to effect digestion of unassociated RNA. 14.The extract of claim 1 which retains immunomodulating activity upontreatment under conditions and for a length of time sufficient to effectdigestion of unassociated proteins.
 15. The extract of claim 1 whichretains immunomodulating activity upon treatment under conditions andfor a length of time sufficient to effect cleavage of glycosidiclinkages with at least one glycosidase selected from the groupconsisting of: amylase, amyloglucosidase, cellulase and neuraminidase.16. The extract of claim 1 which retains immunomodulating activity upontreatment under conditions and for a length of time sufficient to effectcleavage of: (i) three or more α-1,4-linked D-glucose units; (ii)α-1,4-linked glucosides; (iii) α-1,4-linked galactosides; or (iv)α-1,4-linked D-glucose.
 17. The extract of claim 14 wherein thetreatment is digestion with protease.
 18. The extract of claim 1 whereinthe high molecular weight Chlorella polysaccharide and polysaccharidecomplexes are from Chlorella pyrenoidosa.
 19. A pharmaceuticalcomposition comprising the extract of claim 1, in admixture with apharmaceutically acceptable diluent or carrier.
 20. A nutritionalcomposition comprising the extract of claim 1, and at least one energysource selected from the group consisting of carbohydrate, fat andnitrogen sources.
 21. A nutritional supplement comprising the extract ofclaim 1 and a conventional supplement selected from the group consistingof: vitamin E, vitamin C and folic acid.
 22. A nutritional supplementcomprising the extract of claim 1 and a nutraceutical selected from thegroup consisting of: fish oils, spirulina and echinacea.
 23. Acommercial package containing as an active ingredient the extract ofclaim 1, together with instructions for its use as an immunomodulator.24. A process for obtaining a Chlorella extract having immunomodulatingactivity, comprising: (a) size fractionating an aqueous extract ofChlorella, and (b) selecting fractions comprising high molecular weightpolysaccharide and polysaccharide complexes of about 1×10⁵ Da to about1×10⁷ Da.
 25. The process of claim 24 further comprising the step ofpooling and concentrating the selected fractions.
 26. The process ofclaim 24 wherein the size fractionating step comprises chromatography orultrafiltration.
 27. An extract obtained from the process of claim 24.28. A method for modulating the immune response of a mammal, the methodcomprising administering to the mammal an effective amount of theextract of claim
 1. 29. The method of claim 28 wherein modulationcomprises increased proliferation of splenocytes.
 30. The method ofclaim 28 wherein modulation comprises increased production of cytokines.31. The method of claim 30 wherein the cytokine is selected from thegroup consisting of IL-6, IL-10, INF-γ and TNF-α.
 32. A method forsupplementing the immune response to a vaccine in a mammal, the methodcomprising administering an effective amount of the extract of claim 1to the mammal being vaccinated.
 33. The method of claim 32 wherein thevaccine is a flu vaccine.
 34. A method for preventing or treatingbacterial infection in a mammal, the method comprising administering tothe mammal an effective amount of the extract of claim
 1. 35. A methodfor preventing or treating fungal infection in a mammal, the methodcomprising administering to the mammal an effective amount of theextract of claim 1.